The objectives of this proposal are to identify the membrane components of, and to elucidate the regulatory mechanisms controlling, Na+K+2Cl cotransport in avian erythrocytes. This transport system or a close homolog of it, is widespread in the animal kingdom where it plays a central role in the overall control of cellular ionic homeostasis. In order to study Na+K+2Cl cotransport at the molecular level, the avian erythrocyte is a most appropriate model system, in that it is a relatively simple cell that can be obtained in large quantitites as a homogeneous population. Other advantages include the availability of erythrocytes from different adult avian species that exhibit vastly different rates of cotransport, and the existence of embryonic immature red cells that are still actively synthesizing and turning over the cotransport system. The interactions of ions with the cotransporter will be analyzed in transport experiments to ascertain the relationships between the ion binding sites on the molecule. The molecular properties of the Na+K+2Cl cotransporter will be investigated firstly by the interaction of potent inhibitory diuretics with the avian erythrocyte membrane. The characteristics of radiolabeled diuretic binding sites will be defined and compared to similar sites in other tissues. The diuretics will then be chemically modified to produce affinity reagents that may be capable of binding irreversibly to cotransport components. In concert with other labeling techniques, such as lectin- and ATP-binding, these methods should allow us to identify the molecular species involved in cotransport. In avian erythrocytes, Na+K+2Cl cotransport is activated by both cAMP-dependent and -independent stimuli. The cAMP-dependent pathway probably involves phosphorylation of membrane proteins, but the precise mechanism whereby transport function could be regulated by phosphorylation is at present unknown. Identification of cotransport components will allow us to determine whether cyclic nucleotides regulate transport by direct phosphorylation of the transport system or by phosphorylation of other associated membrane proteins. Such information will be applicable to non-erythrocyte tissues, where cyclic nucleotides have also been widely implicated in cotransport regulation. Furthermore, as phosphorylation may be a common mechanism for modulating the activity of a wide variety of transporters, studies of Na+K+2Cl cotransport in the avian erythrocyte will provide essential insights into the general mechanisms whereby such covalent modifications may operate.